The term “species” is understood to mean a chemical species such as a molecule or a complex, or a physical object such as a nanoparticle. The expression “reversibly photoswitchable species” is understood to mean a species that has at least two distinct states having different fluorescence properties and that may be made to pass from one state to the other reversibly under the effect of light. Examples of reversibly photoswitchable fluorescent species are the protein “Dronpa” and the complex “Spinach-DFHBI” (“Spinach” being an RNA aptamer and DFHPI a fluorogenic probe). These species may in particular be used as probes or markers. Other examples of reversibly switchable fluorescent species may be azo derivatives or indeed protein scaffolds.
Fluorescence imaging, and more particularly fluorescence microscopy, has become an indispensable tool in biology, but also in other disciplines such as the science of materials. Its applications are however limited by the ability to observe a signal of interest on a background of fluorescence or noise. This problem is particularly acute in animal or plant in vivo imaging applications, in which the fluorescent markers to be detected are dispersed in a complex autofluorescent and/or scattering medium; the useful signal is then hidden in intense background noise.
Another limit on fluorescence imaging and detecting technique resides in the width of the spectral band of the fluorophores generally employed, with respect to the width of the visible spectral band: it is difficult to selectively detect more than four fluorescent markers in the same sample, because their emission spectra tend to superpose.
To overcome these limits, the patent application WO 2015075209 A1 and the article by J. Querard et al. “Photoswitching Kinetics and Phase-Sensitive Detection Add Discriminative Dimensions for Selective Fluorescence Imaging”, Angew. Chem. Int. Ed. 2015, 54, 266-2637 (2015), disclose a method using reversibly photoswitchable fluorescent probes, in which method a sample, containing a photoswitchable fluorophore species, is illuminated with a periodically modulated light wave. The component of the intensity emitted by the fluorophores at the same angular frequency is then detected, in phase quadrature with respect to the excitation wave. This method allows certain reversibly photoswitchable fluorophores to be selectively detected while minimizing, under certain conditions that are calculated analytically depending on the characteristics of the fluorophore, the noise generated, in conventional methods, by autofluorescence and/or diffusion in the medium of the sample. One of the problems of this method resides in the frequency of acquisition of successive images. The various reversibly photoswitchable fluorescent species used in the prior art are induced to pass from an activated state to their initial non-activated state thermally: the characteristic time of this transition is for example from 5 to 10 seconds and corresponds to the acquisition time of an image using this method. This timescale is too long to take a substantial, biologically relevant number of measurements.
Another prior-art technique is disclosed in the article by Yen-Cheng Chen et al. (Chen, Y. C., Jablonski, A. E., Issaeva, I., Bourassa, D., Hsiang, J. C., Fahmi, C. J., & Dickson, R. M., 2015, Optically Modulated Photoswitchable Fluorescent Proteins Yield Improved Biological Imaging Sensitivity, Journal of the American Chemical Society, 137(40), 12764-12767) which proposes a fluorophore-detecting method that uses two monochromatic sources of laser light of different excitation wavelengths to achieve heterodyne excitation of a reversibly photoswitchable fluorescent species. This technique proposes an empirical choice of the parameters of measurement of the fluorescence of a species, this preventing this type of measurement from being easily transposed to other species. In addition, the signal-to-noise ratio during the measurement of a reversibly photoswitchable fluorescent species is not optimal. Lastly, the disclosed method does not indicate to a person skilled in the art how to observe two reversibly photoswitchable fluorescent species at the same time.
Yet another prior-art technique that makes it possible to exploit the temporal dynamic range of a reversibly photoconvertible probe—which is specific thereto and different from that of interfering fluorophores—to extract a useful signal from the background noise is known as optical lock-in detection (OLID). This technique is described in the article by G. Marriott et al. “Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells”, PNAS, vol. 105, no 46, pages 17789-17794 (18 Nov. 2008), and in the article by Y. Yan et al. “Optical switch probes and optical lock-in detection (OLID) imaging microscopy: high-contrast fluorescence imaging with living systems”, Biochem J (2011), 411-422 and in the article by C. Petchprayoon et al. “Rational design, synthesis, and characterization of highly fluorescent optical switches for high-contrast optical lock-in detection (OLID) imaging microscopy in living cells”, Bioorganic & Medicinal Chemistry 19 (2011), 1030-1040. One drawback of this technique is that it delivers no quantitative information on the concentration of the reversibly photo convertible fluorophores.